Thrombolytic treatment with plasminogen activators (PA) such as tissue plasminogen activator (tPA) has been used not only for treatment of acute myocardial infarction, but also peripheral arterial occlusion (PAO). However, the risk of life-threatening intracranial hemorrhage is measurably high (0.4- 2.9%), particularly for PAO, where treatment can last for 1-2 days. Recent published animal studies have indicated that catheter-administered plasmin (Plm) or its des-kringle derivatives might be more appropriate alternatives to PA, since it has a very short half-life when circulating systemically, and therefore does not result in hemorrhaging. We have produced recombinant miniPlasmin (mPlm) that is proven suitable for treating PAO in animal models. However, our previous results showed that non- specific cleavage at position K698 of mPlm during activation hindered the further development of this promising drug. The overall objective of this proposal is using saturation mutagenesis and process control methods to minimize or eliminate the non-specific cleavage problem and to develop a mutant form of mPlm for PAO treatment. PUBLIC HEALTH RELEVANCE: This project is to develop mutant recombinant miniPlasmin to treat peripheral arterial occlusion (PAO). In addition, the drug may be further developed for treating diseases such as acute ischemic stroke and other blood clotting related illness.